The present invention relates to a confocal microscope and, more particularly, to a confocal microscope which performs excitation and fluorescence observation of a specimen (sample).
As an example of a confocal microscope of this type, for example, a Nipkow disk type confocal microscope using a disk helically formed with a large number of pinholes at equal pitches is known. This Nipkow disk is fabricated such that the respective pinholes are separated from each other by a distance about ten times the pinhole diameter so as not to degrade the image from the sample. Otherwise, crosstalk (light leakage) from each pinhole causes noise in the adjacent pinhole to degrade the quality of the image, and the confocal effect cannot be achieved.
If, however, the space among the pinholes is increased in this manner, the density of the pinholes on the Nipkow disk decreases, and most of light that becomes incident on the disk is undesirably cut by the disk itself. The amount of light passing through the pinholes accordingly becomes, e.g., about one hundredth that of incident light, darkening the image.
In order to solve this drawback, e.g., Jpn. UM Appln. KOKAI Publication No. 4-89906 describes a technique in which microlenses are arranged to correspond to the respective pinholes of the pinhole plate. Although this technique improves the utilization efficiency of the light source, since a large number of lenses are arranged, the pinhole plate becomes expensive.
A technique which is an improvement over the Nipkow disk and pinhole plate described above is described in "Efficient real-time confocal microscopy with white light sources" by R. Juskaitis, T. Wilson, et al., Nature, Vol. 383, October 1996, pp. 804-806. According to this technique, a confocal image having a small degradation can be obtained with a confocal microscope which uses a rotary disk having opening portions and a random pinhole portion where a plurality of pinholes are formed at random.
R. Juskaitis et al. has found that, of two images obtained through the random pinhole portion or opening portions formed in this rotary disk, the image obtained through the random pinhole portion is the sum of a confocal component and a non-confocal component, and the image obtained through one opening portion is a non-confocal component.
FIG. 14 shows the arrangement of a conventional confocal microscope. An optical lens 102 and a half mirror 103 are arranged on the optical path of light emitted by a light source 101, and a sample 106 is arranged on the reflection optical path of the half mirror 103 through a rotary disk 104 and an objective lens 105.
FIG. 15 shows the arrangement of the rotary disk 104. A random pinhole portion 104a on which a plurality of pinholes are formed at random positions and an opening portion 104b through which light can pass freely are formed on the rotary disk 104 at positions to oppose each other. Light-shielding portions 104c and 104d are formed between the random pinhole portion 104a and the opening portion 104b. In the random pinhole portion 104a, the average space among the respective pinholes is substantially equal to the pinhole diameter.
As shown in FIG. 14, the rotary disk 104 is connected to the rotating shaft of a motor (not shown) through a rotating shaft 107, and is rotated at a constant rotation speed. A CCD camera 109 is arranged on the transmission optical path of the half mirror 103 through a condenser lens 108. A computer 110 comprising a CPU and the like is connected to the image output terminal of the CCD camera 109, and a confocal image obtained by arithmetic operation of the computer 110 is displayed on a monitor 111.
With this arrangement, light emitted by the light source 101 passes through the optical lens 102, is reflected by the half mirror 103, and becomes incident on the rotary disk 104 rotating at a constant rotation speed. Light that has passed through the random pinhole portion 104a or opening portion 104b of the rotary disk 104 is focused by the objective lens 105 to become incident on the sample 106. A light beam reflected by the sample 106 when irradiating the sample 106 with light passes through the random pinhole portion 104a or opening portion 104b again, is transmitted through the half mirror 103, and is focused on the image pickup surface of the CCD camera 109 by the condenser lens 108. The image pickup timing of the CCD camera 109 is controlled in synchronism with the rotation speed of the rotary disk 104, and the CCD camera 109 separately picks up two images that have passed through the random pinhole portion 104a and opening portion 104b.
More specifically, when light emitted by the light source 101 passes through the random pinhole portion 104a, and is focused by the objective lens 105 to become incident on the sample 106, the light beam reflected by the sample 106 at this time passes through the random pinhole portion 104a again, is transmitted through the half mirror 103, and is focused by the condenser lens 108 to form an image, including a confocal component and a non-confocal component, on the image pickup surface of the CCD camera 109. The image picked up by the CCD camera 109 and including the confocal and non-confocal components is fetched by the computer 110 as an image signal and is accumulated as image data.
When light emitted by the light source 101 passes through the opening portion 104b and is focused by the objective lens 105 to become incident on the sample 106, the light beam reflected by the sample 106 at this time passes through the opening portion 104b again, is transmitted through the half mirror 103, and is focused by the condenser lens 108 to form an image including the non-confocal component on the image pickup surface of the CCD camera 109. The image including the non-confocal image picked up by the CCD camera 109 is fetched by the computer 110 as an image signal and is accumulated as image data.
In this manner, of the two images obtained through the random pinhole portion 104a and opening portion 104b of the rotary disk 104, the image obtained through the random pinhole portion 104a is the sum of the confocal component and the non-confocal component, and the image obtained through the opening portion 104b is the non-confocal component. Hence, the computer 110 obtains a confocal image by calculating a difference between the image obtained through the random pinhole portion 104a and the image obtained through the opening portion 104b, and displays this confocal image on the monitor 111. The stereoscopic image near the surface of the sample 106 is obtained by moving the sample 106 in a vertical direction a shown in FIG. 14 by, e.g., a piezoelectric element or a movable table.
In the conventional Nipkow disk type confocal microscope, reflected light that can be utilized is 0.5% to 1% the incident light emitted by the light source. However, it is reported that, with the confocal microscope of T. Wilson et al., reflected light that can be utilized is 25% to 50% the incident light emitted by the light source, so that a brighter image can be obtained.
FIG. 16 shows the arrangement of a conventional Nipkow disk type scanning confocal microscope. This confocal microscope can perform excitation and fluorescence observation of the specimen, and is constituted by a light source 201, a scanner unit 202 having a lens 223, an excitation filter 203A, a microlens disk 204, a short-path dichroic mirror 205, and a pinhole disk 206, an objective lens 208, and an observation unit 210 having a photometric filter 211, a condenser lens 212, and an image pickup element 213.
Referring to FIG. 16, light emitted by the light source 201 is collimated into parallel light by the lens 223, and this parallel light passes through the excitation filter 203A and is transmitted through the microlenses of the microlens disk 204. The parallel light which has been transmitted through the microlenses is then transmitted through the dichroic mirror 205 and is focused on the pinhole surface formed on the pinhole disk 206. The light which has passed through the pinholes of the pinhole disk 206 is transmitted through the objective lens 208 and is focused again on the surface of a specimen 209. Thereafter, the fluorescence emitted by the surface of the specimen 209 is transmitted through the objective lens 208 and focused on the pinhole surface. The light which has been transmitted through the pinholes is reflected by the dichroic mirror 205, passes through the photometric filter 211 and condenser lens 212, is focused on the image pickup element 213 or an eyepiece (not shown), and is confirmed as a specimen image.
The excitation filter 203A for extracting excitation light is set between the light source 201 and microlens disk 204, and the photometric filter 211 is set before the image pickup element 213 or eyepiece (not shown). This enables 1-wavelength excitation and 1-wavelength fluorescence observation.
With the arrangement shown in FIG. 16, however, both the excitation filter 203A and photometric filter 211 for transmitting a specific wavelength are fixed. Although 1-wavelength excitation and 1-wavelength fluorescence observation and measurement can be performed, multiple excitation and multiple fluorescence observation and measurement (to be described hereinbelow) are sometimes difficult to perform depending on the type of the specimen. Multiple excitation is excitation of a specimen with a plurality of wavelengths when introducing excitation light. Multiple fluorescence observation is observation and measurement of fluorescence, emitted from a specimen, with a plurality of wavelengths.
FIG. 17 shows the arrangement of a scanning type confocal microscope in which a dichroic mirror 331 and observation units 330A and 330B are added to the arrangement of FIG. 16 in order to improve the drawbacks of the arrangement shown in FIG. 16. The observation unit 330A is constituted by a condenser lens 312A, a photometric filter 328A, and a TV (television) camera 329A. The observation unit 330B is constituted by a condenser lens 312B, a photometric filter 328B, and a TV (television) camera 329B.
In the arrangement shown in FIG. 17, since the observation units 330A and 330B are disposed on the optical path of the dichroic mirror 331, 2-wavelength fluorescence observation and measurement can be performed if the photometric filters 328A and 328B have different wavelengths. When a ratio image (a ratioing image) is to be calculated based on the respective images obtained by the TV cameras 329A and 329B, it is very difficult to perform adjustment such that the pixels of the images of the respective TV cameras 329A and 329B coincide with each other. Since the performances of the two TV cameras 329A and 329B vary, when the TV cameras 329A and 329B are to be replaced with new ones, adjustment must always be performed, which is cumbersome.